Anticoagulant reversal agents

ABSTRACT

Novel anticoagulant reversal compounds are disclosed, as well as methods of making the compounds, pharmaceutical compositions including the compounds, methods of using the compounds to reverse the anticoagulant effects of coagulation inhibitors, and diagnostic assays comprising the compounds.

This application claims the benefit of priority from U.S. ProvisionalPatent Application No. 61/564,559, which was filed on Nov. 29, 2011,U.S. Provisional Patent Application No. 61/614,292, which was filed onMar. 22, 2012, U.S. Provisional Patent Application No. 61/641,698, whichwas filed on May 2, 2012, and U.S. Provisional Patent Application No.61/666,291, which was filed on Jun. 29, 2012, the contents of which areincorporated herein in their entirety.

FIELD OF THE INVENTION

The present invention discloses compounds that completely or partiallyreverse anticoagulant effects of coagulation inhibitors, such asunfractionated heparin (“UFH”), low molecular weight heparin (“LMWH”),fondaparinux, and other antithrombin binding anticoagulants, as well asdirect Xa and Ha inhibitors.

BACKGROUND OF THE INVENTION

The coagulation cascade is a normal physiological process which aims atpreventing significant blood loss or hemorrhage following vascularinjury. There are times, however, when a blood clot (thrombus) will formwhen it is not needed. For instance, some high risk conditions such asacute medical illness, prolonged immobilization, surgery, or cancer canincrease the risk of developing a blood clot which can potentially leadto significant consequences such as atherosclerotic cardiovasculardisease and/or abnormal cardiac rhythms.

The coagulation cascade consists of a series of steps in which aprotease cleaves and subsequently activates the next protease in thesequence. Each protease can activate several molecules of the nextprotease in the series, amplifying this biological cascade. The finalresult of these reactions is to convert fibrinogen, a soluble protein,to insoluble threads of fibrin. Together with platelets, the fibrinthreads form a stable blood clot.

Antithrombin (AT), a serine protease inhibitor, is the major plasmainhibitor of coagulation proteases. AT blocks the coagulation cascadeby, e.g., inhibiting thrombin (factor IIa) and activated factor X(factor Xa). Heparin (unfractionated heparin) and low molecular weightheparins (LMWHs; fractionated heparin) inhibit the coagulation processthrough binding to AT via a pentasaccharide sequence. This binding leadsto a conformational change of AT, which accelerates its inhibition offactors IIa, Xa, and other proteases involved in blood clotting. Oncedissociated, heparin and LMWH are free to bind to another antithrombinmolecule and subsequently inhibit more thrombin and factor Xa.

Unfractionated heparin is a mixture of glycosaminoglycans (GAGs)discovered in the liver of dogs to have anti-coagulant properties in1916 by McLean and Howell at Johns Hopkins University. In addition toanti-coagulation, unfractionated heparin has been found to have otherproperties including anti-inflammation and angiogenesis. LMWHs areheparins consisting of short chains of polysaccharide, generally havingmolecular weight of less than 8000 Da. LMWH and heparin are both used toprevent blood from clotting inside the body, but are used in differentsituations in the clinic.

Heparin is available as a liquid solution administered parenterally.LMWH, such as enoxaparin, is a low molecular weight fraction of heparin.It is also available as a liquid injectable solution. The currentlyavailable brands of LMWH approved by FDA in the United States areLOVENOX® (generic name, enoxaparin) and FRAGMIN® (generic name,dalteparin).

Low molecular weight or fractionated heparin has greater specificity forblood factor Xa and factor IIa activity than unfractionated heparin.Additionally, LMWH has a more reproducible effect on activated partialthromboplastin time (aPTT), a measure of coagulation time. LWMH has alower incidence of Heparin Induced Thrombocytopenia (HIT). Because LMWHhas more predictable efficacy and a lower incidence of adverse effectssuch as HIT, patients can inject LMWH themselves at home, although it isalso often used in the hospital. For these reasons, LMWHs have becomethe market-leading anticoagulant.

Protamine, a positively charged molecule, can be used to reverseanti-coagulation resulting from administration of highly negativelycharged unfractionated heparin or low molecular weight heparin (LMWH).Protamine is a natural product that has been associated with supplyproblems, which highlights a need for additional, ideally synthetic,reversal agent options. The anti-coagulant activity of LMWH can bepartially, but not fully, reversed by intravenous administration ofprotamine. The reason for the reduced anticoagulation reversal activityof protamine in the case of LMWH is believed to be due to a lesserbinding affinity for the LMWH fraction in the blood than unfractionatedheparin. Protamine must be administered slowly, due to hypotensiveeffects and concerns regarding anaphylaxis.

Recently, additional anticoagulant agents have begun to gain regulatoryapproval. Examples of such anticoagulants include dabigatran orPRADAXA®, argatroban or ARGATROBAN®, rivaroxaban or XARELTO®, apixabanor ELIQUIS®, edoxaban or LIXIANA®, and fondaparinux or ARIXTRA®. Theseanticoagulants inhibit either factor IIa or factor Xa from propagatingcoagulation.

Anticoagulants such as dabigatran, fondaparinux, rivaroxaban andapixaban have no approved reversal agent. The current state of the artfor dabigatran or PRADAXA® reversal is to employ activated charcoal toattempt to remove dabigatran from the blood and to use bloodtransfusions. Other than Eerenberg et al. Circulation. 2011 Oct. 4;124(14):1573-9. Epub 2011 Sep. 6, which reports that in a small clinicaltrial, prothrombin complex concentrate was able to reverse dabigatran,but not rivaroxaban, there is no data or clinically available antidotefor reversing any of these coagulation Factor IIa or Xa inhibitors.Therefore, when patients are anti-coagulated with these agents, adverseeffects associated with overdosing, particularly significant or fatalbleeds, are much more dangerous than the side effects associated withadministration of unfractionated heparin. The lack of reversal agenttherefore limits the use of these drugs.

For these reasons, there is a longstanding, strong, unmet clinical needfor new anti-coagulation reversal agents.

SUMMARY OF THE INVENTION

Inhibitors of heparin, heparin fragments, fondaparinux and other factorXa or factor IIa inhibitors has been developed. The general structure ofthe anti-coagulant reversal agents of interest is: R-Z-R′, where R andR′ are positively charged agents at physiologic pH and can be the sameor different molecules and Z is a hydrophobic cyclic or fused ringcompound. More specifically, the inhibitor is represented by a compoundof formula I or a pharmaceutically acceptable salt thereof:

Y-M-X-L-A-L′-X′-M′-Y′  (I)

wherein:

A is a substituted or unsubstituted aromatic or non-aromatic,carbocyclic or heterocyclic ring or a linear moiety;

L and L′ are the same or different and are linkers;

X and X′ are the same or different and are absent or are a functionalgroup that attaches the linker L to M and the linker L′ to M′,respectively;

M and M′ are the same or different and are absent or is a linker thatattaches X to Y and X′ to Y′, respectively; and

Y and Y′ are the same or different and are a moiety containing one ormore cationic atoms or groups or one or more groups that become cationicunder physiological conditions.

The compounds can be symmetrical or asymmetrical; that is, one or moreof L, L′, X, X′, M, M′, Y, or Y′ can be the same or different. Thecompounds can be chiral (i.e., contain one or more chiral centers) orachiral.

In some embodiments, A is a heterocyclic moiety. In other embodiments, Ais a heterocyclic moiety and L and L′ are a substituted or unsubstitutedalkylene chain. In still other embodiments, A is a heterocyclic moiety,L and L′ are a substituted or unsubstituted alkylene chain, and X and X′are —NH—C(═O)—. In still other embodiments, A is a heterocyclic moiety,L and L′ are a substituted or unsubstituted alkylene chain, X and X′ are—NH—C(═O)—, and M and M′ are a substituted alkylene chain. In stillother embodiments, A is a heterocyclic moiety, L and L′ are asubstituted or unsubstituted alkylene chain, X is —NH—C(═O)—, M and M′are a substituted alkylene chain, and Y and Y′ are a guanidine moiety.In particular embodiments, A is a 1,4 or 2,5 disubstituted piperazinering.

In another embodiment of the invention the inhibitor is a compoundrepresented by the formula II or a pharmaceutically acceptable saltthereof:

wherein each of L, L′, M, M′, Y and Y′ are as described herein.

In another embodiment of the invention the inhibitor is a compoundrepresented by the formula III or a pharmaceutically acceptable saltthereof:

wherein L, L′, M, M′, Y and Y′ are as described herein.

In yet another embodiment of the invention the inhibitor is a compoundrepresented by the formula (IV) or pharmaceutically acceptable saltthereof:

wherein Y and Y′ are as described herein and n is 3 to 5, m is 3 to 6and G is selected from —NH₂ and OH. Most preferably, G is amino.

Yet another embodiment of the invention the inhibitor is a compoundrepresented by any of formula II, III or IV and Y and Y′ areindependently selected from the group consisting of

Most preferably G is —NH₂ and Y and Y′ are

In the preferred embodiment, the compound is di-arginine piperazine(DAP), depicted in formula V, or a related compound, depicted in formulaVI, or pharmaceutically acceptable salts of either compound:

-   2-Amino-5-guanidino-pentanoic acid    (3-{4-[3-(2-amino-5-guanidino-pentanoylamino)-propyl]-piperazin-1-yl}-propyl)-amide;    or

-   2-Amino-5-guanidino-pentanoic acid    {5-[(2-amino-5-guanidino-pentanoylamino)-methyl]-piperazin-2-ylmethyl}-amide.

In a specific embodiment, the compound of formula V is a stereoisomer asdepicted in formula VII:

In another specific embodiment, the compound of formula VI is astereoisomer as depicted in formula VIII:

The compounds of the invention can be administered in a pharmaceuticalcomposition as an aqueous solution as a bolus and/or intravenousinfusion, subcutaneous injection, or orally. In the preferredembodiment, the compounds are administered by injection (intravenous,intramuscular or subcutaneous) in a carrier such as distilled sterilewater, saline, buffered saline, or another pharmaceutically acceptableexcipient for injection. In some embodiments, the inhibitor may beadministered orally, to a mucosal surface (nasal, pulmonary, vaginal,rectal or buccal) or by depot.

The compounds of the invention may be administered in pharmaceuticalcomposition to the patient in need of reversal of heparin, LMWH or otherthrombin inhibitor-mediated anticoagulation in an effective amount torestore normal coagulation and hemostasis. The pharmaceuticalcompositions including the compound of the inventions are suitable forhospital use or in non-emergency home reversal. It is administered tothe patient in need of reversal of heparin, LMWH or other thrombininhibitor mediated anticoagulation in an effective amount to restorecoagulation. The compounds and pharmaceutical compositions describedherein can also be used to reduce the activity of heparin-binding growthfactors and/or for reversing completely or in part a combination of oneor more Factor IIa and/or Factor Xa anticoagulant agents.

Thus, the compounds of the invention can be used in a method ofcompletely or partially reversing an anticoagulant effect of acoagulation inhibitor. The compounds of the invention can also be usedas a part of a diagnostic kit, e.g., a diagnostic kit for determiningconcentration of an anticoagulant in the blood.

Examples demonstrate that DAP directly bound rivaroxaban, apixaban,unfractionated heparin, fondaparinux, and LMWH, reversing anticoagulantactivity. DAP reversed oral rivaroxaban and subcutaneous LMWHanticoagulation in vivo as measured by aPTT and subcutaneousfondaparinux as measured by Xa activity in rats. DAP reversal, confirmedby statistically significant reduction in blood loss in tail rattransection assay, was shown for apixaban, dabigatran, edoxaban, andrivaroxaban. DAP completely reversed apixaban and rivaroxaban at a dosemass ratio of about 10:1 DAP:anticoagulant in human blood ex vivo asmeasured using an anti-Xa kit. DAP exhibited a dose-dependent reversalof apixaban and rivaroxaban in human blood ex vivo. Rivaroxaban reversalin freshly drawn human whole blood was confirmed by aPTT measurements exvivo. DAP did not bind argatroban concentrations up to 1:1000 in vitro.DAP reversed oral dabigatran in vivo in rats as measured by aPTT.Argatroban dosed rats remained anticoagulated after a 200× IV dose ofDAP, showing that DAP is safe and that the reversal interaction isspecific for the heparins and new oral anticoagulants. In summary, theexamples demonstrate complexation of DAP to heparin and LMWH and thatDAP serves as an excellent reversal agent for heparin, heparin-likecompounds and other thrombin inhibitors including dabigatran, approvedlow molecular weight heparins, as well as rivaroxaban (XARELTO®),fondaparinux (ARIXTRA®), edoxaban (LIXIANA®), and apixaban (ELIQUIS®),as tested in in vitro assays with human blood, anti-Xa and aPTT testsand/or in vivo in a rat tail transection assay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of heat flow versus temperature as measured bydifferential scanning calorimetry (DCS) in which DAP is heated from −20°C. to 200° C. (“1” or “first heat”), cooled to −20° C., and heated backto 200° C. (“2” or “second heat”).

FIG. 2 is a graph of DAP alone, UFH alone, and DAP-UFH combination, as afunction of volume percent compared to size (d·nm) as measured byDynamic Light Scattering (DLS).

FIG. 3 is a graph of DAP alone, rivaroxaban alone and DAP-rivaroxaban inratios of 1:1 and 10:1, DAP:rivaroxaban, as a function of volume(percent) compared to size (d·nm) as measured by DLS.

FIG. 4 is a graph of DAP alone, apixaban alone and DAP-apixaban bindingin ratios of 1:1, 10:1 and 100:1, as a function of volume (percent)compared to size (d·nm) as measured by DLS.

FIG. 5 is a graph of DAP alone, fondaparinux alone and DAP-fondaparinuxbinding in ratios of 1:1, 10:1 and 100:1, as a function of volume(percent) compared to size (d·nm) as measured by DLS.

FIG. 6 is a graph of DAP alone, LMWH alone and DAP-LMWH binding inratios of 1:1, 1:10 and 100:1, as a function of volume (percent)compared to size (d·nm) as measured by DLS.

FIG. 7 is a graph of DAP alone, argatroban alone and DAP-argatrobanbinding in ratios of 1:1, 10:1, 100:1, and 1000:1, as a function ofvolume (percent) compared to size (d·nm) as measured by DLS.

FIG. 8 is a graph of activated partial thromboplastin time (aPTT,seconds) measured over time (hours) during five hours after subcutaneousadministration of 10 mg of bemiparin (LMWH) to a rat. Four hours intotreatment, the rat received an intravenous dose of 200 mg/kg (100 mg)DAP.

FIG. 9 is a graph of activated partial thromboplastin time (aPTT,seconds) measured over time (hours) after oral administration ofPRADAXA® (dabigatran) to a rat followed by intravenous administration of200 and 100 mg/kg (100 mg and 50 mg) DAP.

FIG. 10 is a graph of activated partial thromboplastin time (aPTT)measured over time (hours) after subcutaneous administration ofunfractionated heparin (UFH) to a rat followed by intravenousadministration of 200 mg/kg (100 mg) and 400 mg/kg (200 mg) DAP.

FIG. 11 is a graph of aPTT (seconds) measured over time (hours) afteroral administration of 5 mg/kg of rivaroxaban to a rat followed byintravenous administration of 5 mg/kg (2 mg) DAP.

FIG. 12 is a graph of active fondaparinux concentration (ng/mL) measuredover time (minutes after reversal) after a subcutaneous administrationof 5 mg/kg fondaparinux to a rat, followed by intravenous administrationof 200 mg/kg DAP (i.e., “reversal”).

FIG. 13 is a graph of aPTT (seconds) measured over time (minutes afterreversal) after oral administration of 15.5 mg/kg PRADAXA® (dabigatran)to a rat, followed by intravenous administration of 100 mg/kg DAP (i.e.,“reversal”).

FIG. 14 is a graph of the aPTT time (seconds) for 0, 2, 10, 25, 50, and100 mg intravenously administered DAP.

FIG. 15 is a graph of blood collected (i.e., cumulative blood loss) over30 minutes in a rat tail transection bleeding assay in rats receiving 2mg rivaroxaban and 0 mg DAP, 2 mg rivaroxaban and 2.5 mg DAP, 2 mgrivaroxaban and 12.5 mg DAP, or sham reversal and anticoagulant doses(“sham”). With groups of three age-matched rats, 12.5 mg of DAP reducedblood loss to sham dose levels yielding a statistically significantdifference (*p<0.05) from rats receiving rivaroxaban only.

FIG. 16 is a graph of blood collected (i.e., cumulative blood loss) over30 minutes in a rat tail transection bleeding assay in rats receiving1.25 mg apixaban and 0 mg DAP, 1.25 mg apixaban and 5 mg DAP, 1.25 mgapixaban and 12.5 mg DAP, or sham reversal and anticoagulant doses(“sham”). With groups of three age-matched rats, 5 mg and 12.5 mg of DAPreduced blood loss to sham dose levels yielding a statisticallysignificant difference (***p<0.01) from rats receiving apixaban only.

FIG. 17 is a graph of blood collected (i.e., cumulative blood loss) over30 minutes in a rat tail transection bleeding assay in rats receiving1.25 mg edoxaban and 0 mg DAP, 1.25 mg edoxaban and 12.5 mg DAP, or shamreversal and anticoagulant doses (“sham”). With groups of threeage-matched rats, 12.5 mg of DAP reduced blood loss to sham dose levelsyielding a statistically significant difference (*p<0.05) from ratsreceiving edoxaban only.

FIG. 18 is a graph of blood collected (i.e., cumulative blood loss) over30 minutes in a rat tail transection bleeding assay in rats receiving 15mg dabigatran etexilate and 0 mg DAP, 15 mg dabigatran etexilate and 5mg DAP, 15 mg dabigatran etexilate and 12.5 mg DAP, or sham reversal andanticoagulant doses (“sham”). With groups of three age-matched rats,12.5 mg of DAP reduced blood loss to sham dose levels yielding astatistically significant difference (***p<0.01) from rats receivingdabigatran etexilate only.

FIG. 19 is a graph of aPTT (seconds) measured in freshly drawn humanblood treated ex vivo with 50 micrograms/ml DAP, 0.25 micrograms/mlrivaroxaban, 50 micrograms/ml DAP and 0.25 micrograms/ml rivaroxaban, orsaline.

FIG. 20 is a graph showing effective anticoagulant concentrationmeasured by anti-factor Xa activity assay in human plasma treated exvivo with of 218 ng/L rivaroxaban alone or in combination with 1,250mg/L DAP, and 459 ng/L rivaroxaban alone or in combination with 6,250ng/L DAP.

FIG. 21 is a graph showing effective anticoagulant concentrationmeasured by anti-factor Xa activity assay in human plasma treated exvivo with 156 ng/L apixaban alone or in combination with 1,156 ng/L DAP,and 313 ng/L apixaban alone or in combination with 3,125 ng/L DAP.

FIG. 22 is a graph showing effective anticoagulant concentrationmeasured by anti-factor Xa activity assay in human plasma treated exvivo with 218 ng/L rivaroxaban, alone or in combination with increasingamounts (1.25, 12.5, 125, and 1,250 ng/L) of DAP.

DETAILED DESCRIPTION OF THE INVENTION I. Anticoagulant Reversal Agents

Novel anticoagulant reversal agents are disclosed. The compounds of theinvention include compounds described herein, as well as thepharmaceutically acceptable salts thereof.

Inhibitors of heparin, heparin fragments, fondaparinux and factor Xa orfactor IIa inhibitors (e.g., oral factor Xa or factor IIa inhibitors)have been developed. The general structure of the anti-coagulantreversal agents of interest is: R-Z-R′, where R and R′ are positivelycharged agents at physiologic pH and can be the same or differentmolecules and Z is a hydrophobic cyclic or fused ring compound.

More specifically, the inhibitor is a compound of the formula (I) orpharmaceutically acceptable salt thereof:

Y-M-X-L-A-L′-X′-M′-Y′  (I)

wherein:

A is a substituted or unsubstituted aromatic or non-aromatic,carbocyclic or heterocyclic ring or a linear moiety;

L and L′ are the same or different and are linkers;

X and X′ are the same or different and are absent or are a functionalgroup that attaches the linker L to M and the linker L′ to M′,respectively;

M and M′ are the same or different and are absent or is a linker thatattaches X to Y and X′ to Y′, respectively; and

Y and Y′ are the same or different and are a moiety containing one ormore cationic atoms or groups or one or more groups that become cationicunder physiological conditions.

The compounds can be symmetrical or asymmetrical; that is, one or moreof L, L′, X, X′, M, M′, Y, or Y′ can be the same or different. Thecompounds can be chiral (i.e., contain one or more chiral centers) orachiral.

In some embodiments, A is a heterocyclic moiety. In other embodiments, Ais a heterocyclic moiety and L and L′ are a substituted or unsubstitutedalkylene chain. In still other embodiments, A is a heterocyclic moiety,L and L′ are a substituted or unsubstituted alkylene chain, and X and X′are —NH—C(═O)—. In still other embodiments, A is a heterocyclic moiety,L and L′ are a substituted or unsubstituted alkylene chain, X and X′ are—NH—C(═O)—, and M and M′ are a substituted alkylene chain. As usedherein, alkylene chain is a divalent alkelene moiety that is C₁ to C₁₀,preferably C₃ to C₆ in length, and which may be substituted orunsubstituted. Exemplary substituents include alkyl, hydroxyl, hydroxylalkyl, amino, amino alkyl, alkoxy, alkyl alkoxy. As used herein, theterm alkyl is C₁ to C₁₀, preferably C₁-C₆ straight chain or branchedhydrocarbon. In still other embodiments, A is a heterocyclic moiety, Land L′ are a substituted or unsubstituted alkylene chain, X is—NH—C(═O)—, M and M′ are a substituted alkylene chain, and Y and Y′ area guanidine moiety.

In some embodiments, A is a non-aromatic, heterocyclic ring, such aspiperazine or diketopiperazine. In other embodiments, A is a linearmoiety, such as a linear diamine or other linear moiety containingreactive functional groups that can form a bond to X and X′, whenpresent, or Y and Y′. In some embodiments, the linkers L and L′ areattached to the heteroatoms in the ring A, such as the two nitrogenatoms in piperazine. In other embodiments, the linker L and L′ areattached to atoms other than the heteroatoms in the ring, such ascarbon. In particular embodiments, A is a 1,4 or 2,5 disubstitutedpiperazine ring. In some embodiments, L and L′ and/or M and M′ are asubstituted or unsubstituted alkylene chains, such as —(CH₂)_(n)—, wheren is an integer from 1-10, preferably from 1-6, e.g., 1-3. In particularembodiments, n is 3. In some embodiments, L and/or M are absent.

X and X′ are a functional group that attaches the linkers L and L′ to Yand Y′. Exemplary functional groups include, but are not limited to,esters, amides, carbonates, and ketones. In particular embodiments, Xand X′ are a functional group that is resistant to simple hydrolysis,such as an amide group.

Y and Y′ are a moiety that contains one or more atoms or groups that arecationic or will be cationic under physiological conditions. Examplesinclude amine and guanidine moieties as well as phosphorous containingmoieties, such as alkyltriphenylphosphonium, tetraphenylphosphonium,tetraphenylarsonium, tribenzyl ammonium, and phosphonium moieties.Additional cationic moieties include cationic oligomers and polymers,such as oligo- or polylysine, oligo- or polyarginine, N-alkylatedpolyethylene imine, and the like. Other cationic moieties includedelocalized lipophilic cations containing one to three carbimino,sulfimino, or phosphinimino units as described in Kolomeitsev et al.,Tet. Let., Vol. 44, No. 33, 5795-5798 (2003).

In some embodiments, the compound is a piperazine derivative, whereinthe amino acid side chains contain one or more positively charged atomsor atoms that will be positively charged under physiological conditions.Examples include diarginine piperazine. Other amino acids that arepositively charged or will be positively charged under physiologicalconditions can be substituted for arginine.

“Aromatic”, as used herein, refers to 5-12-membered, preferably 5-, 6-and 7-membered aromatic, heterocyclic, fused aromatic, fusedheterocyclic, biaromatic, or bihetereocyclic ring systems, optionallysubstituted. Broadly defined, “Ar”, as used herein, includes 5-, 6- and7-membered single-ring aromatic groups that may include from zero tofour heteroatoms, for example, benzene, pyrrole, furan, thiophene,imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine,pyridazine and pyrimidine, and the like. Those aryl groups havingheteroatoms in the ring structure may also be referred to as “arylheterocycles” or “heteroaromatics”. The aromatic ring can be substitutedat one or more ring positions with such substituents as described above,for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromaticor heteroaromatic moieties, —CF₃, —CN, or the like. The term “Ar” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (i.e.,“fused rings”) wherein at least one of the rings is aromatic, e.g., theother cyclic ring or rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls and/or heterocycles. Examples of heterocyclic ringinclude, but are not limited to, benzimidazolyl, benzofuranyl,benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl,benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl,carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl,furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl,indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl,isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl,isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl,morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl,phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl,phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl,piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl,pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl and xanthenyl.

“Heterocycle” or “heterocyclic”, as used herein, refers to a cyclicradical attached via a ring carbon or nitrogen of a monocyclic orbicyclic ring containing 3-10 ring atoms, and preferably from 5-6 ringatoms, consisting of carbon and one to four heteroatoms each selectedfrom the group consisting of non-peroxide oxygen, sulfur, and N(R)wherein R is absent or is H, O, (C₁₋₄)alkyl, phenyl or benzyl, andoptionally containing 1-3 double bonds and optionally substituted withone or more substituents. Examples of heterocyclic ring include, but arenot limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl.

In another embodiment of the invention the inhibitor is a compoundrepresented by the formula II or a pharmaceutically acceptable saltthereof:

wherein each of L, L′, M, M′, Y and Y′ are as previously described.

In another embodiment of the invention the inhibitor is a compoundrepresented by the formula III or a pharmaceutically acceptable saltthereof:

wherein L, L′, M, M′, Y and Y′ are as previously described.

In yet another embodiment of the invention the inhibitor is a compoundrepresented by the formula (IV) or pharmaceutically acceptable saltthereof:

wherein Y and Y′ are as previously described and n is 3 to 5, m is 3 to6 and G is selected from —NH₂ and OH. Most preferably, G is amino.

Yet another embodiment of the invention the inhibitor is a compoundrepresented by any of formula II, III or IV and Y and Y′ areindependently selected from the group consisting of

Most preferably G is —NH₂ and Y and Y′ are

Thus, in one embodiment, the compound of the invention is di-argininepiperazine (“DAP”), such as the compound of formula V, or a relatedcompound of formula VI, or pharmaceutically acceptable salts of eithercompound:

-   2-Amino-5-guanidino-pentanoic acid    (3-{4-[3-(2-amino-5-guanidino-pentanoylamino)-propyl]-piperazin-1-yl}-propyl)-amide;    or

-   2-Amino-5-guanidino-pentanoic acid    {5-[(2-amino-5-guanidino-pentanoylamino)-methyl]-piperazin-2-ylmethyl}-amide.

In a specific embodiment, the compound of formula V is a stereoisomer asdepicted in formula VII:

In another specific embodiment, the compound of formula VI is astereoisomer as depicted in formula VIII:

The phrase “pharmaceutically acceptable salt” of a compound as usedherein means a salt that is pharmaceutically acceptable and thatpossesses the desired pharmacological activity of the parent compound.Pharmaceutically acceptable salts include salts of acidic or basicgroups present in compounds of the invention. Pharmaceuticallyacceptable acid addition salts include, but are not limited to,hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate,phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate,citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate,maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzensulfonate, p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Suitable base saltsinclude, but are not limited to, aluminum, calcium, lithium, magnesium,potassium, sodium, zinc, and diethanolamine salts.

The compound of the invention inhibits activity of coagulationinhibitors. One proposed mechanism of action of the compound of theinvention is through binding negatively charged molecules (e.g.,fondaparinux, unfractionated heparin, LMWH, described herein). Othercoagulation inhibitors (e.g., factor IIa and factor Xa inhibitors suchas dabigatran, apixaban, edoxaban, and rivaroxaban, described herein)also possess negative charges; thus, the compound of the invention mayinhibit these coagulation inhibitors through neutralization of theirnegatively charged moieties.

Another proposed mechanism of action of the compound of the invention isthrough weak physical interactions such as hydrogen bonding andhydrophobic interactions with the coagulation inhibitors. Oral FactorIIa and Xa inhibitors possess hydrophobic portions, which may causehydrophobic association with the compound of the invention, e.g., DAP.

Thus, in some embodiments, the compounds of the invention contain atleast one cyclic hydrophobic moiety, e.g., one or a combination ofaliphatic or aromatic rings including fused rings. In other embodiments,the compounds of the invention contain at least one cyclic hydrophobicmoiety and a least two positively charged or partially charged moietiesat physiological pH.

In some embodiments of the invention, one or both arginines of thecompounds of Formulas V and VI (or the compounds of Formulas VII andVIII) are substituted by one or more positively charged amino acids,their derivatives, or similarly charged compounds, e.g., lysine,histidine, ornithine. The arginines in the compounds of Formulas V andVI or positively charged amino acids substituted for such arginines canbe naturally occurring amino acids (i.e., L-amino acids), theirenantiomers (i.e., D-amino acids), or racemic or other mixtures thereof“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L or R and Sare used to denote the absolute configuration of the molecule about itschiral center(s). The prefixes D and L or (+) and (−) are employed todesignate the sign of rotation of plane-polarized light by the compound,with (−) or L meaning that the compound is levorotatory. A compoundprefixed with (+) or D is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

In other embodiments of the invention, the compound of the inventioncontains at least one cyclic hydrophobic moiety, e.g., one or acombination of aliphatic and aromatic rings including fused rings.Compounds of interest contain at least one cyclic hydrophobic moiety andat least two positively charged or partially charged moieties atphysiological pH.

Special consideration should be given to the design of peptide-basedtherapeutic agents, since such agents may cause unwanted and oftensevere immunological reactions once administered to a subject. Thecompound of the invention is designed to be of sufficiently lowmolecular weight to minimize immunogenicity issues. In one embodiment,in order to avoid activation of the immune response, the compound isdesigned such that its molecular weight is less than about 5000 daltons,such as less than or about 1000 daltons, e.g., about 500 daltons. In oneembodiment, the molecular weight of the compound is about 512 daltons.

It is preferable that the compounds of the invention do not bind, orotherwise interfere with the function of the ERG, a potassium ionchannel that contributes to the electrical conductivity of the heart.Inhibition of this potassium channel may lead to potentially fatal longQT syndrome, and some otherwise successful drug candidates haveexhibited human ERG binding.

In addition, it is preferable that the compound of the invention doesnot inhibit or serve as substrates for membrane-bound cytochrome p450(CYP) enzymes. CYPs are major enzymes involved in drug metabolism, andmodulation of CYP activity may interfere with clearance and metabolismof other drugs administered to a subject, causing unwanted druginteractions.

Also preferably, the compounds of the invention do not exhibitsignificant plasma protein binding in vitro (e.g., albumin binding).Because the compounds of the invention are largely unbound to plasmaproteins, they exhibit short activity half-lives minimizing the risk ofaccumulation-based overdose.

II. Synthesis of Anticoagulant Reversal Agents

The compounds and their pharmaceutically acceptable salts describedherein are prepared using a variety of methods starting fromcommercially available compounds, known compounds, or compounds preparedby known methods. Exemplary synthetic routes to one of the compoundsdescribed herein (Compound of Formula V, di-arginine piperazine, “DAP”)are included in the schemes below. The schemes below are also applicableto the DAP stereoisomer compound of Formula VII by selecting theappropriate stereoisomeric starting compounds. Other compounds of theinvention may be synthesized following a similar synthetic scheme. It isunderstood by those skilled in the art that the order of steps shownherein may be changed to accommodate functionality in the targetmolecule. It is also understood by those skilled in the art that variousprotection and deprotection steps may be required for synthesis. Theneed for protection and deprotection, and the selection of appropriateprotecting groups are found, for example, in Greene and Wuts, ProtectingGroups in Organic Synthesis, Second Edition, John Wiley & Sons (1991),which is incorporated herein by reference in its entirety.

In some embodiments of the present invention, the protecting group istertiary butyloxycarbonyl group (Boc). In other embodiments of thepresent invention, the protecting group is2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl group (Pbf). Inanother embodiment, amino acid protecting group may be, but is notlimited to, 2,2,5,7,8-pentamethyl-chroman-6-sulphonyl (PMC).

Protecting groups may be removed by a variety of routes. Removal ofprotecting group comprises, e.g., treating protected compound withtrifluoroacetic acid (TFA), aqueous HCl, or heating in acetic acid.Because removal of protecting groups, e.g., removal of protecting groupsunder acidic conditions, can result in production of cationic speciesthat can alkylate the functional groups on the peptide chain, scavengersmay be added during the deprotection step to react with any of the freereactive species. Examples of scavengers include, but are not limitedto, water, anisol derivatives and thiol derivatives. Thus, in oneembodiment, removal of protecting groups comprises treating protectedcompound with TFA and a scavenger (e.g., TFA and water).

Various solvents, e.g., organic solvents, may be used in the steps ofthe synthesis. Appropriate solvents include, but are not limited to,dimethyl sulfoxide, dimethylformamide (DMF), tetrahydrofuran, methanol,ethanol, methylene chloride, toluene, and acetone. In some embodiments,the solvent is DMF.

Suitable acid binding agents may be used in the steps of the synthesis.These include, but are not limited to, organic bases, such as, forexample, pyridine, triethylamine, triethanolamine,1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and diisopropylethylamine(DIEA); and inorganic bases, such as, for example, sodium hydride,potassium carbonate, and sodium carbonates. In some embodiments, theacid binding agent is DIEA.

Synthesis may include peptide coupling reagents. Peptide couplingreagents may include, but are not limited to,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC),N-Hydroxybenzotriazole (HOBt), carbonyldiimidazole (CDI),dicyclohexylcarbodiimide (DCC), active N-hydroxysuccinamide (OSu) ester,O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU), and combinations thereof. In one embodiment, the peptidecoupling reagent is HBTU. In another embodiment, the peptide couplingreagent is EDC/HOBt. In yet another embodiment, the peptide couplingreagent is an active OSu ester.

Additionally, the synthesis may include a step in which a crude productis purified, e.g., by column chromatography. The desired products ofeach step or series of steps may be separated and/or purified to thedesired degree of homogeneity by the techniques common in the art.Typically such separations involve multiphase extraction,crystallization from a solvent or solvent mixture, distillation,sublimation, or chromatography. Chromatography can involve any number ofmethods including, for example: reverse-phase and normal phase; sizeexclusion; ion exchange; high, medium, and low pressure liquidchromatography methods and apparatus; small scale analytical; simulatedmoving bed (SMB) and preparative thin or thick layer chromatography, aswell as techniques of small scale thin layer and flash chromatography.

In one scheme, the compound of Formula V (DAP)

is synthesized by reacting excess equivalents (e.g., at least about twoequivalents) of compound 1

with one equivalent of compound 2

in the presence of a peptide coupling reagent, to obtain a compound 3

wherein P1 is a protecting group and P2 is a protecting group or is ahydrogen.

In one embodiment, the peptide coupling reagent is HBTU, EDC/HOBt, or anactive OSu ester. In one embodiment, the protecting group P1 is Boc. Inanother embodiment, the protecting group P2 is Pbf. In a differentembodiment, the protecting group P1 is Boc and P2 is a hydrogen.

Subsequently, 3 may be purified. This purification may involve variouscolumn chromatography methods known in the art.

Protecting groups of 3 may be removed by a variety of methods known inthe art in order to obtain the compound of Formula V. Deprotection canbe achieved by, e.g., removal of protecting groups using trifluoroaceticacid (TFA) and water, TFA and water or another scavenger, including, butnot limited to aqueous HCl, or heating in acetic acid.

The compound may be further purified using a column chromatographymethod, such as ion exchange chromatography with salt buffers orpreparative HPLC with trifluoroacetic acid or acetic acid as a buffer.

In a more specific scheme, the coupling involved reacting compound 1,wherein P1 was Boc and P2 was a hydrogen (depicted as Boc-Arg-OH.HClbelow), with compound 2 as depicted below:

The resultant crude product was more than 95% pure by thin layerchromatography (TLC).

Subsequently, the deprotection step was carried out as depicted below:

The deprotected product was purified by preparative HPLC using 1% aceticacid buffer. Product purity of >98% was observed. Residual TFA wasremoved by low quantity of DOWEX resin. The molecular weight of DAP (thecompound of Formula V) is 512.4, and the compound synthesized accordingto the above scheme exhibited the following primary peak by massspectroscopy: [M+H]⁺=513.4.

III. Pharmaceutical Compositions

Pharmaceutical compositions comprising the compounds described hereinare provided. Such a composition may contain, in addition to thecompound of the invention, a pharmaceutically acceptable carrier orexcipient. The term “pharmaceutically acceptable” means a nontoxicmaterial that is compatible with the physical and chemicalcharacteristics of the active ingredient and does not interfere with theeffectiveness of the biological activity of the active. The compositionsmay contain various diluents, fillers, salts, buffers, stabilizers,solubilizers, and other materials well known in the art. Thecharacteristics of the carrier will depend on the route ofadministration, and are generally well known in the art.

The pharmaceutical composition of the invention may be adapted forenteral administration—administration of the composition, wherein thecomposition is absorbed through the digestive tract, e.g., oralingestion, rectal administration. In other embodiments, thepharmaceutical composition of the invention may be adapted forparenteral administration—administration of the composition, wherein thecomposition is introduced via a route other than digestive tract, e.g.,intravenous, subcutaneous, cutaneous, nasal, pulmonary, vaginal, buccalroute.

Suitable pharmaceutical compositions, e.g., compositions for oraladministration, may be prepared as described in references such as“Pharmaceutical dosage form tablets”, eds. Liberman et. al. (New York,Marcel Dekker, Inc., 1989), “Remington—The science and practice ofpharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, Md.,2000, and “Pharmaceutical dosage forms and drug delivery systems”,6^(th) Edition, Ansel et. al., (Media, Pa.: Williams and Wilkins, 1995),incorporated herein by reference, which provide information on carriers,materials (e.g., coating materials), equipment and process for preparingtablets and capsules and delayed release dosage forms of tablets,capsules, and granules.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate; polyvinylacetate phthalate, acrylic acid polymers and copolymers, and methacrylicresins that are commercially available under the trade name Eudragit®(Roth Pharma, Westerstadt, Germany), Zein, shellac, and polysaccharides.Additionally, the coating material may contain conventional carrierssuch as plasticizers, pigments, colorants, glidants, stabilizationagents, pore formers and surfactants.

Optional pharmaceutically acceptable excipients present in thedrug-containing tablets, beads, granules or particles include, but arenot limited to, diluents, binders, lubricants, disintegrants, colorants,stabilizers, and surfactants.

Diluents, also termed “fillers,” are typically necessary to increase thebulk of a solid dosage form so that a practical size is provided forcompression of tablets or formation of beads and granules. Suitablediluents include, but are not limited to, dicalcium phosphate dihydrate,calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose,microcrystalline cellulose, kaolin, sodium chloride, dry starch,hydrolyzed starches, pregelatinized starch, silicone dioxide, titaniumoxide, magnesium aluminum silicate and powder sugar.

Binders are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (Polyplasdone XL from GAF ChemicalCorp).

Stabilizers are used to inhibit or retard drug decomposition reactionswhich include, by way of example, oxidative reactions.

Surfactants may be anionic, cationic, amphoteric or nonionic surfaceactive agents. Suitable anionic surfactants include, but are not limitedto, those containing carboxylate, sulfonate and sulfate ions. Examplesof anionic surfactants include sodium, potassium, ammonium of long chainalkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodiumN-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate,myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

Pharmaceutical compositions of the invention may be designed to providedelayed, sustained, pulsatile or other modified release.

If desired, the tablets, beads granules or particles may also containminor amount of nontoxic auxiliary substances such as wetting oremulsifying agents, dyes, pH buffering agents, and preservatives.

Bioadhesive formulations may also be utilized to enhance uptake ormodify release. Such formulations are known in the art. See, forexample, US Patent Application No. 20060045865 by Jacob, incorporatedherein by reference.

Pharmaceutical compositions adapted for delivery via nasal or pulmonaryadministration may also be useful. Aerosols for the delivery oftherapeutic agents to the respiratory tract have been described, forexample, Adjei, A. and Garren, J. Pharm. Res., 7: 565-569 (1990); andZanen, P. and Lamm, J.-W. J. Int. J. Pharm., 114: 111-115 (1995). Therespiratory tract encompasses the upper airways, including theoropharynx and larynx, followed by the lower airways, which include thetrachea followed by bifurcations into the bronchi and bronchioli. Theupper and lower airways are called the conducting airways. The terminalbronchioli then divide into respiratory bronchioli which then lead tothe ultimate respiratory zone, the alveoli, or deep lung. Gonda, I.“Aerosols for delivery of therapeutic and diagnostic agents to therespiratory tract,” in Critical Reviews in Therapeutic Drug CarrierSystems, 6:273-313 (1990). The deep lung, or alveoli, is the primarytarget of inhaled therapeutic aerosols for systemic drug delivery.

Drugs administered by inhalation may come as liquid aerosolformulations.

For injectable compositions (e.g., intravenous compositions), thecarrier is distilled sterile water, saline, buffered saline, or anotherpharmaceutically acceptable excipient for injection. Additives mayinclude preservatives and acids or base to adjust pH, to altersolubility or uptake.

In one embodiment, wherein the pharmaceutical composition comprises theDAP compound of formula V (or its stereoisomer of formula VII) and thecomposition is adapted for parenteral administration in an injection,the compound is dissolved in water with appropriate tonicity andmolality modifiers (such as phosphate buffered saline). DAP iswater-soluble at greater than 100 mg/ml. In the one embodiment, DAP isadapted as a sterile solution for IV administration. In one aspect, themolality of the pharmaceutical composition in which DAP is adapted forIV administration is adjusted to 290 mOsm/L with sodium chloride, andthe pH is adjusted to 7.4 with sodium hydroxide. Preferably thepharmaceutical composition is administered as an intravenous bolus byslow push.

IV. Methods of Use

The present invention provides a method of completely or partiallyreversing an anticoagulant effect of a coagulation inhibitor comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound of the invention (e.g., a compound of formula I,II, III, IV, V, VI, VII, or VIII) or pharmaceutically acceptable saltthereof. The present invention also provides a method of promotingcoagulation in a subject in need thereof, wherein the subject isreceiving a coagulation inhibitor, comprising administering to thesubject a therapeutically effective amount of a compound of theinvention or a pharmaceutically acceptable salt thereof. In addition,the present invention provides a method of neutralizing or inhibiting acoagulation inhibitor comprising administering to a subject in needthereof a therapeutically effective amount of a compound of theinvention or a pharmaceutically acceptable salt thereof.

In the present invention, coagulation inhibitor (also referred to hereinas anticoagulant) is a molecule that inhibits coagulation process.Exemplary coagulation inhibitors include, but are not limited to,antithrombin activators (e.g., unfractionated heparin and LMWH), factorIIa inhibitors, and factor Xa inhibitors.

Heparin: Heparin is a naturally occurring mucopolysaccharide that actsin the body as an antithrombin co-factor to prevent intravascularclotting. The substance is produced by basophils and mast cells, whichare found in large numbers in the connective tissue surroundingcapillaries, particularly in the lungs and liver. In the form of sodiumsalt, heparin is used therapeutically as an anticoagulant.

Low Molecular Weight Heparin: Low Molecular Weight Heparin (LMWH) ismade from heparin using various methods of depolymerization, includingoxidative depolymerization with hydrogen peroxide, used in themanufacture of ardeparin (NORMIFLO®); deaminative cleavage with isoamylnitrite, used in the manufacture of certoparin (SANDOPARIN®); alkalinebeta-eliminative cleavage of the benzyl ester of heparin, used in themanufacture of enoxaparin (LOVENOX® and CLEXANE®); oxidativedepolymerization with Cu²⁺ and hydrogen peroxide, used in themanufacture of parnaparin (FLUXUM®); beta-eliminative cleavage by theheparinase enzyme, used in the manufacture of tinzaparin (INNOHEP® andLOGIPARIN®); deaminative cleavage with nitrous acid, used in themanufacture of dalteparin (FRAGMIN®), reviparin (CLIVARIN®) andnadroparin (FRAXIPARIN®), which results in the formation of an unnaturalanhydromannose residue at the reducing terminal of the oligosaccharidesproduced. This can subsequently be converted to anhydromannitol using asuitable reducing agent. Both chemical and enzymatic beta-eliminationresult in the formation of an unnatural unsaturated uronate residue (UA)at the non-reducing terminal.

Summary of anticoagulant activities of several LMWHs is presented inTable 1.

TABLE 1 Molecular weight (MW) data and anticoagulant activities ofcurrently available LMWH products. Ratio anti-Xa/ LMWH Average molecularweight anti-IIa activity BEMIPARIN 3600 9.7 CERTOPARIN 5400 2.4DALTEPARIN 6000 2.5 ENOXAPARIN 4500 3.9 NADROPARIN 4300 3.3 PARNAPARIN5000 2.3 REVIPARIN 4400 4.2 TINZAPARIN 6500 1.6 Adapted from Gray E. etal., Thromb Haemost, 99: 807-818 (2008).

Clinically, LMWH (average molecular weight of about 4.5 kDa) differsfrom heparin (i.e., “unfractioned heparin”; average molecular weight ofabout 15 kDa) in a variety of ways: (a) LMWH requires less frequentsubcutaneous dosing for postoperative prophylaxis of venousthromboembolism; (2) LMWH requires once or twice daily subcutaneousinjection in patients treated for venous thromboembolism and unstableangina instead of intravenous infusion required with heparin; (3) LMWHrequires no monitoring of the aPTT coagulation parameter; (4) LMWH posesa lower risk of bleeding; (5) long term use of LMWH poses a lower riskof osteoporosis; and (6) LMWH poses a lower risk of heparin-inducedthrombocytopenia (a potential side effect of heparin administration).However, the anticoagulant effects of heparin are typically reversiblewith protamine sulfate, while protamine's effect on LMWH is limited. Inaddition, LMWH has less effect on thrombin (Factor IIa) activitycompared to heparin, while both LMWH and heparin have a similar effecton Factor Xa activity.

Thrombin and Other Factor IIa or Xa Inhibitors: Examples of thrombin(Factor IIa) and factor Xa inhibitors include anticoagulants such asdabigatran (PRADAXA®), rivaroxaban (XARELTO®), apixaban (ELIQUIS®),edoxaban (LIXIANA®), fondaparinux (ARIXTRA®), and argatroban(ARGATROBAN®).

The chemical name for oral anticoagulant PRADAXA®, dabigatran etexilatemesylate, a direct thrombin inhibitor, is β-Alanine,N-[[2-[[[4-[[[(hexyloxy)carbonyl]amino]iminomethyl]phenyl]amino]methyl]-1-methyl-1H-benzimidazol-5-yl]carbonyl]-N-2-pyridinyl-,ethylester, methanesulfonate. Dabigatran and its acyl glucuronides arecompetitive, direct thrombin inhibitors. Because thrombin (Factor IIa,serine protease) enables the conversion of fibrinogen into fibrin duringthe coagulation cascade, its inhibition prevents the development of athrombus.

Rivaroxaban, a factor Xa inhibitor, is the active ingredient inXARELTO®, and has the chemical name5-Chloro-N-({(5S)-2-oxo-3-[4-(3-oxo-4-morpholinyl)phenyl]-1,3-oxazolidin-5-yl}methyl)-2-thiophenecarboxamide.Rivaroxaban is a pure (S)-enantiomer. XARELTO® is an orally bioavailablefactor Xa inhibitor that selectively blocks the active site of factor Xaand does not require a cofactor (such as Anti-thrombin III) foractivity.

Apixaban or ELIQUIS® is1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-4,5-dihydropyrazolo[5,4-c]pyridine-3-carboxamide.It is an orally administered direct factor Xa inhibitor approved inEurope and presently undergoing phase III trials in the U.S. for theprevention of venous thromboembolism.

Edoxaban or LIXIANA® isN′-(5-chloropyridin-2-yl)-N-[(1S,2R,4S)-4-(dimethylcarbamoyl)-2-[(5-methyl-6,7-dihydro-4H-[1,3]thiazolo[5,4-c]pyridine-2-carbonyl)amino]cyclohexyl]oxamide.Edoxaban is a direct factor Xa inhibitor, and it has been approved inJapan for use in preventing venous thromboembolism.

ARIXTRA® is fondaparinux sodium. It is a synthetic and specificinhibitor of activated Factor X (Xa). Fondaparinux sodium is methylO-2-deoxy-6-O-sulfo-2-(sulfoamino)-α-D-glucopyranosyl-(1→4)-O-β-D-glucopyranuronosyl-(1→4)-O-2-deoxy-3,6-di-O-sulfo-2-(sulfoamino)-α-D-glucopyranosyl-(1→4)-O-2-O-sulfo-α-L-idopyranuronosyl-(1→4)-2-deoxy-6-O-sulfo-2-(sulfoamino)-α-D-glucopyranoside,decasodium salt. The molecular formula of fondaparinux sodium isC₃₁H₄₃N₃Na₁₀O₄₉S₈ and its molecular weight is 1728. The structuralformula is provided below:

The antithrombotic activity of fondaparinux sodium is the result ofantithrombin III (ATIII)-mediated selective inhibition of Factor Xa. Byselectively binding to ATIII, fondaparinux sodium potentiates (about 300times) the innate neutralization of Factor Xa by ATIII. Neutralizationof Factor Xa interrupts the blood coagulation cascade and thus inhibitsthrombin formation and thrombus development. Fondaparinux sodium doesnot inactivate thrombin (activated Factor II) and has no known effect onplatelet function. At the recommended dose, fondaparinux sodium does notaffect fibrinolytic activity or bleeding time. Thepharmacodynamics/pharmacokinetics of fondaparinux sodium are derivedfrom fondaparinux plasma concentrations quantified via anti-factor Xaactivity. Only fondaparinux can be used to calibrate the anti-Xa assay.(The international standards of heparin or LMWH are not appropriate forthis use.) As a result, the activity of fondaparinux sodium is expressedas milligrams (mg) of the fondaparinux calibrator. The anti-Xa activityof the drug increases with increasing drug concentration, reachingmaximum values in approximately three hours. Fondaparinux sodiumadministered by subcutaneous injection is rapidly and completelyabsorbed (absolute bioavailability is 100%). In patients undergoingtreatment with fondaparinux sodium injection 2.5 mg, once daily, thepeak steady-state plasma concentration is, on average, 0.39 to 0.50 mg/Land is reached approximately 3 hours post-dose. In these patients, theminimum steady-state plasma concentration is 0.14 to 0.19 mg/L. Inpatients with symptomatic deep vein thrombosis and pulmonary embolismundergoing treatment with fondaparinux sodium injection 5 mg (bodyweight <50 kg), 7.5 mg (body weight 50 to 100 kg), and 10 mg (bodyweight >100 kg) once daily, the body-weight-adjusted doses providesimilar mean steady-state peaks and minimum plasma concentrations acrossall body weight categories. The mean peak steady-state plasmaconcentration is in the range of 1.20 to 1.26 mg/L. In these patients,the mean minimum steady-state plasma concentration is in the range of0.46 to 0.62 mg/L.

ARGATROBAN® is a synthetic direct thrombin (Factor IIa) inhibitor,derived from L-arginine. The chemical name for ARGATROBAN® is1-[5-[(aminoiminomethyl)amino]-1-oxo-2-[[(1,2,3,4-tetrahydro-3-methyl-8-quinolinyl)sulfonyl]amino]pentyl]-4-methyl-2-piperidinecarboxylicacid, monohydrate. The molecular formula of ARGATROBAN® isC₂₃H₃₆N₆O₅S.H₂O. Its molecular weight is 526.66. ARGATROBAN® is a directthrombin inhibitor that reversibly binds to the thrombin active site.ARGATROBAN® does not require the co-factor antithrombin III forantithrombotic activity. ARGATROBAN® is administered by injection, andit exerts its anticoagulant effects by inhibiting thrombin-catalyzed orthrombin-induced reactions, including fibrin formation; activation ofcoagulation factors V, VIII, and XIII; activation of protein C; andplatelet aggregation.

An anticoagulant effect is any effect of a coagulation inhibitor (e.g.,heparin, LMWH, Factor Xa inhibitor, Factor IIa inhibitor) that resultsfrom its blockage of the propagation of the coagulation cascades.Nonlimiting examples of anticoagulation effects include upregulation ofantithrombin activity, decreased Factor Xa activity, decreased FactorIIa activity, increased blood loss, and any other conditions wherein theactivity or concentrations of clotting factors are altered in such a wayas to inhibit blood clot formation.

Activity of a coagulation inhibitor (i.e., its anticoagulant effects)may be measured by a variety of methods, including but not limited to achromogenic anti-factor Xa activity assay, activated partialthromboplastin time assay, prothrombin time, bleeding assay (e.g., rattail bleeding assay), thromboelastography, thrombin generation assay,dilute Russel's viper venom time, ecarin clotting time, kaolin clottingtime, International Normalized Ratio (INR), fibrinogen testing (Clauss),thrombin time (TCT), mixing time, and euglobulin lysis time. Thesemethods aid in determining various anticoagulation parameters, and areknown to those skilled in the art. Thus, in some embodiments,anticoagulation can be monitored by one or a combination of the abovelisted assays.

The anti-factor Xa assay directly measures anti-factor Xa activity. Themethodology behind an anti-factor Xa assay is that patient plasma isadded to a known amount of excess factor Xa and excess antithrombin. Ifa factor Xa inhibitor is present in the patient plasma, it will reducethe enzymatic activity of factor Xa. The amount of residual factor Xa isinversely proportional to the amount of anti-Xa agent in the plasma. Theamount of residual factor Xa is detected by adding a chromogenicsubstrate that mimics the natural substrate of factor Xa, makingresidual factor Xa cleave it, releasing a colored compound that can bedetected by a spectrophotometer. Antithrombin deficiencies in thepatient do not affect the assay, because excess amounts of antithrombinare provided in the reaction. Results are given in anticoagulantconcentration in units/mL of antifactor Xa, such that high valuesindicate high levels of anticoagulation and low values indicate lowlevels of anticoagulation.

The activated partial thromboplastin time (aPTT) assay is an assay thatmeasures how long it takes for the blood to clot. Blood samples arecollected for direct measurement or in tubes with oxalate and citrate toarrest coagulation by calcium until the assay can be performed. In theassay, a phospholipid, an activator (silica, celite, kaolin, ellagicacid, etc.), and calcium are mixed into the plasma to inducecoagulation. The assay measures the time until a thrombus (clot) forms.

Rat tail bleeding assay or rat tail transection assay is an assay thatmeasures blood loss, e.g., blood loss after drug administration. In oneembodiment, where the effect of the compound of the invention (e.g.,DAP) is being tested, at the Tmax of the anticoagulant, DAP isadministered intravenously. After 20 minutes, rat tails are transectedapproximately 1 mm from the tip, placed in room temperature saline, andblood is collected for 30 minutes and weighed.

Assays used to measure activity of coagulation inhibitors maybe used inthe laboratory or in the clinic to measure reversal of an anticoagulanteffect of a coagulation inhibitor, e.g., reversal of an anticoagulanteffect of a coagulation inhibitor due to administration of apharmaceutical composition comprising a compound of the invention. Thus,in one embodiment, the assays are utilized to measure complete orpartial reversal of an anticoagulant effect of a coagulation inhibitor(such as heparin, LMWH, Factor IIa inhibitor, and Factor Xa inhibitor).

A complete reversal of an anticoagulant effect of a coagulationinhibitor occurs upon neutralization of the anticoagulant activity. Inone embodiment, a complete reversal of an anticoagulant effect of acoagulation inhibitor, as measured by the anti-Xa activity assay, occurswhen anticoagulant concentration is brought below the minimum effectiveconcentration (MEC) for anticoagulation. MEC, as used herein, is alowest amount of the drug (e.g., coagulation inhibitor) required fortherapeutic effect. In another embodiment, a complete reversal of ananticoagulant effect of a coagulation inhibitor, as measured by the aPTTassay, occurs when the aPTT returns within about 10% of baseline. Abaseline, as used herein, refers to aPTT in the absence of coagulationinhibitors.

In many cases, anticoagulation will still be desired, but to a lesserdegree. Thus, a partial reversal of an anticoagulant effect of acoagulation inhibitor will be indicated. Partial reversal of ananticoagulant effect of a coagulation inhibitor, as measured by theanti-Xa activity assay, occurs when the anticoagulant concentration isbrought below the anticoagulant concentration in the absence of ananticoagulation reversal agent (e.g., a compound of the invention), butremains above the MEC for anticoagulation. Thus, in some embodiments,partial reversal of an anticoagulation effect of coagulation inhibitorsoccurs when the concentration of anticoagulant is lower than about fourtimes the MEC, preferably about twice the MEC, more preferably less thanabout twice the MEC (e.g., at about the MEC). Partial reversal of ananticoagulant effect of coagulation inhibitor, as measured by aPTTassay, occurs when aPPT is reduced below the measurement in the absenceof an anticoagulation reversal agent (e.g., a compound of the invention)but above the baseline. Thus, in other embodiments, partial reversal ofan anticoagulation effect of coagulation inhibitors occurs when the aPTTmeasurement is reduced below about four times the baseline, preferablyabout twice the baseline, more preferably less than about twice thebaseline. Generally, the extent and duration of anticoagulation reversalis determined by the physician or veterinarian.

As used herein, “subject in need thereof” is a subject in need of eitheracute or planned reversal of anticoagulation, e.g., a subject sufferingfrom anticoagulant overdose, a subject suffering from hemorrhage (e.g.,trauma-induced hemorrhage or spontaneous hemorrhage in the GI tract orelsewhere), a subject requiring planned surgical intervention, a subjectundergoing an invasive or non-invasive procedure requiring a biopsy, asubject undergoing a procedure wherein a procedural error may riskhemorrhage if the subject remains anticoagulated, a subject requiringspinal or epidural anesthesia. “Subject in need thereof” may be apatient in whom the presence of a direct factor inhibitor (Factor Xa,Factor IIa and/or antithrombin) is producing or is likely to producebleeding effects. Thus, “subject in need thereof” may be a subjectreceiving anticoagulation therapy (e.g., subject receiving heparin,LMWH, Factor IIa inhibitor, or Factor Xa inhibitor) for, e.g., strokeprevention, cardiac surgical and diagnostic procedures, cardiacarrhythmias, deep vein thrombosis (DVT) prevention, pulmonary embolism,general prevention of the formation of pathologic blood clots.

“Subject in need thereof,” as used herein, is an animal. “Subject inneed thereof” includes, without limitation, a human, mouse, rat, guineapig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesusmonkey. In one embodiment, “subject in need thereof” is a mammal. Inanother embodiment, “subject in need thereof” is a human.

As used herein, “therapeutically effective amount” refers to an amountof an anticoagulation reversal agent (e.g., a compound of the inventiondescribed herein), which is effective, upon single or multiple doseadministration (e.g., bolus and/or maintenance doses) to a subject, inneutralizing or inhibiting (completely or partially reversing) ananticoagulant effect of a coagulation inhibitor or in promotingcoagulation.

In one aspect, a therapeutically effective amount is a dose of ananticoagulation reversal agent that is between 0.01 and 10,000 times theanticoagulant dose by weight. In another aspect, the anticoagulationreversal agent is administered at a dose mass ratio of between about 1:1and 1000:1 of the anticoagulation reversal agent to anticoagulant, e.g.,100:1 of the anticoagulation reversal agent to anticoagulant, such as10:1 of anticoagulation reversal agent to anticoagulant. In oneembodiment of the present method, a therapeutically effective amount ofthe anticoagulation reversal agent may be administered by subcutaneous,intramuscular, or intravenous route of administration. For example, itmay be administered intravenously as a sterile solution. In anotherembodiment, a therapeutically effective amount of the anticoagulationreversal agent is administered by oral, nasal, or pulmonary route, or toa mucosal region (mouth, rectum, or vagina).

The therapeutically effective amount of the anticoagulation reversalagent (i.e., the compound of the invention) will typically range fromabout 0.001 mg/kg to about 1 g/kg of body weight per day; in anotherembodiment, from about 0.01 mg/kg to about 600 mg/kg body weight perday; in another embodiment, from about 0.01 mg/kg to about 250 mg/kgbody weight per day; in another embodiment, from about 0.01 mg/kg toabout 400 mg/kg body weight per day; in another embodiment, from about0.01 mg/kg to about 200 mg/kg of body weight per day; in anotherembodiment, from about 0.01 mg/kg to about 100 mg/kg of body weight perday; in one embodiment, from about 0.01 mg/kg to about 25 mg/kg bodyweight per day; in another embodiment, from about 0.1 mg/kg to about 10mg/kg body weight per day; in another embodiment, from about 0.001 mg/kgto about 100 mg/kg of body weight per day; in another embodiment, fromabout 0.001 mg/kg to about 10 mg/kg of body weight per day; and inanother embodiment, from about 0.001 mg/kg to about 1 mg/kg of bodyweight per day. Standard coagulation assays (as those described herein)and other in vitro assays can be used to determine the therapeuticallyeffective amount.

In some aspects of the invention, the compound of the invention may beco-administered with at least one additional therapeutic agent. In oneembodiment, the at least one additional therapeutic agent may be vitaminK, which is typically used to correct clotting deficiencies induced bywarfarin compounds.

The present invention also provides a diagnostic assay for determiningthe anticoagulant concentration in the blood. As shown in Example 13below, DAP demonstrates a dose-responsive trend in reversing rivaroxabanex vivo in human plasma using a 510 k-cleared anti-factor Xa chromogenicassay. Thus, the compound of the invention, e.g., DAP, can be used in adiagnostic assay to determine the concentration of an anticoagulant,e.g., a Factor Xa inhibitor, in the blood. In such an assay, thecompound of the invention, e.g., DAP, can be used either in conjunctionwith the currently available kit reagents or as a direct bindingsubstrate replacing synthetic factors present in currently availablekits. In one embodiment, the diagnostic assay may comprise the compoundof the invention (e.g., DAP) as a binding substrate, wherein thecompound of the invention binds an anticoagulant in a blood sample, andthe residual activity of the clotting factor (e.g., Factor Xa) isquantified to determine the concentration of the anticoagulant in thesample. In another embodiment, the diagnostic assay may comprise thecompound of the invention (e.g., DAP) conjugated to magneticmicroparticles, wherein the compound of the invention can bind ananticoagulant in a blood sample in order to either remove theanticoagulant from the sample or to concentrate it. The DAP-basedchromogenic or point of care assay of the invention can aid in thedetermination of anticoagulant levels in subjects, which is currently asignificant clinical unmet need since current diagnostics cannotdetermine blood concentrations of direct inhibitors with high accuracy.

Additionally, the present invention provides an assay, e.g., achromogenic assay, to determine the concentration of the compound of theinvention, e.g., DAP, required to reverse the anticoagulant present inthe blood. In one embodiment, the assay uses DAP as a direct bindingagent for various anticoagulants.

The invention also provides an assay, e.g., a chromogenic assay, todetermine the amount of the compound of the invention, e.g., DAP, in theblood. Such assay may utilize predetermined concentrations of ananticoagulant.

The present invention also provides a diagnostic kit that incorporates adiagnostic assay described herein above. Thus, in one embodiment, thekit is used for determining the anticoagulant concentration in theblood. The kit may contain other components, packaging, instructions,reagents, and/or other material to aid in the determination ofanticoagulant (e.g., Factor Xa inhibitor) or DAP concentration and toaid in the use of the kit. Additionally, the kit may be used todetermine if there is a combination of warfarin and anotheranticoagulant as warfarin will be unaffected by the compound of theinvention, while other anticoagulants will be reversed.

As demonstrated in the following examples, a compound of the invention(e.g., DAP) is capable of binding heparin, inactivating it in vivo.Thus, in addition to its effects on coagulation, a compound of theinvention may also be used to deprive tissues of the biochemicalactivities of heparin. For example, other heparin-binding molecules havedemonstrated the ability to reduce fibroblast growth factor (FGF),epidermal growth factor (EGF), vascular endothelial growth factor(VEGF), and other heparin binding growth factors. VEGF and FGFdeprivation has been shown useful in anti-cancer therapy, makingcompounds of the invention possible candidates for the treatment ofcancer. Therefore, in one aspect, the present invention provides amethod for treating, preventing, or ameliorating a cancer in a subject,comprising administering to a subject a therapeutically effective amountof a compound of the invention or a pharmaceutically acceptable saltthereof.

As demonstrated in the examples, one compound of the invention, DAP,bound XARELTO®, ELIQUIS®, ARIXTRA® and LMWH in vitro as measured bydynamic light scattering (DLS). DAP reversed subcutaneously administeredARIXTRA® and LMWH in vivo. DAP reversed XARELTO®, ELIQUIS®, PRADAXA®,LIXIANA®, unfractionated heparin and bemiparin in vivo. DAPintravenously administered at 100 mg/kg, 250 mg/kg and 400 mg/kg dosesin rats showed no adverse effect. DAP was orally bioavailable in rats.DAP exhibited no human ERG binding, did not inhibit or serve as asubstrate of CYP enzymes, and did not appreciably bind any plasmaproteins (data not shown). In addition, it appears that DAP has a shortelimination half-life, because anti-coagulation induced by PRADAXA®returned in 20-30 minutes following an intravenous bolus dose of DAP inrats. Moreover, DAP was stable to sterilization (survived heating to200° C.) and to storage as a lyophilized powder at 4° C. for more thanone year. Summary of anticoagulant reversal by DAP is presented in Table2.

TABLE 2 Anticoagulant reversal Trade Generic Blood Factor Route of BindsReversal Name Name Company Inhibited Administration DAP Agents Lovenox ®Enoxaparin Sanofi, Sandoz/ ~80-90% Xa, s.c. Injection X ProtamineHibor ® Bemiparin Momenta, Rovi ~10-20% IIa * & DAP Arixtra ®Fondaparinux GSK Xa s.c. Injection X DAP Eliquis ® Apixaban Pfizer, XaOral X DAP BMS Xarelto ® Rivaroxaban Bayer, Xa Oral X DAP Janssen, J&JArgatroban ® Argatroban GSK IIa s.c. Injection — None Pradaxa ®Dabigatran Boehringer IIa Oral X DAP etexilate Ingelheim * Protaminepartially reverses low molecular weight heparins.

TABLE 3 In vitro in vivo correlation DLS Binding Reversal Blood DrugGeneric Molar Ratio Molar Ratio in vivo Factor(s) Route of Name[DAP/drug] [DAP/drug] Measure Inhibited Administration Rivaroxaban 9  3* Bleeding assay Xa Oral Apixaban 10   8* Bleeding assay Xa OralFondaparinux 3 130 Xa kit Xa s.c. Injection Bemiparin 7 140 aPTT ~80-90%Xa, s.c. Injection ~10-20% IIa Argatroban N/A N/A aPTT IIa s.c.InjectionAssumes oral bioavailabilities of 60% for rivaroxaban, 50% for apixaban,and 5% for dabigatran; Assumes 100% bioavailability for injectableanticoagulants.

Summary of in vitro-in vivo correlation of treatment with DAP ispresented in Table 3. DLS binding molar ratio is calculated by dividingthe lowest mass ratio of DAP to anticoagulant that shows significantbinding, defined as an association in phosphate buffered saline above 50nm in apparent diameter, by the molecular weight ratio of DAP and theanticoagulant. The molecular weights used in the calculations were 512Da (DAP), 436 Da (rivaroxaban), 460 Da (apixaban), 1.7 kDa(fondaparinux), 3.6 kDa (bemiparin), 628 Da (dabigatran), and 509 Da(ARGATROBAN®). Reversal molar ratio was calculated similarly using theminimal in vivo reversal dose of DAP necessary to achieve reversal asmeasured by the rat tail transection bleeding assay, chromogenic anti-Xakit, or by activated partial thromboplastin time (aPTT). For thebleeding assay, the anticoagulant was considered reversed if the bloodloss over a period of 30 minutes after tail transection, with the cuttail immersed in room temperature saline, was within 25% of the control(no anticoagulant administered). As measured by the Xa kit, reversal wasachieved when the effective anticoagulant concentration was broughtbelow the minimum effective concentration (MEC) for anticoagulation. Asmeasured by aPTT, reversal was considered achieved when ananticoagulated rat aPTT returned to within 10% of baseline. In the caseof fondaparinux, although 200 mg/kg DAP was the lowest dose administeredin vivo, the in vitro data indicate that significantly lower reversaldoses are possible.

The entire contents of all references, patent applications, and patentscited throughout this application are hereby incorporated by referenceherein.

EXAMPLES

The invention will be further illustrated in the following nonlimitingExamples. These Examples are set forth to aid in the understanding ofthe invention but are not intended to, and should not be construed to,limit its scope in any way. The Examples do not include detaileddescriptions of conventional methods that are well known to those ofordinary skill in the art.

Example 1: In Vitro Stability Testing of Diarginine Piperazine (“DAP”)Materials and Methods

An acetate salt of DAP was prepared as described herein. As described inthese examples, DAP solid or powder refers to the acetate salt, whileDAP in solution refers to the free base as the salt ionizes in aqueoussolution. As described in these examples, the DAP compound used was thecompound of Formula VII.

The DAP powder was tested for thermal stability in two ways. DAP wasstored at 4° C. for 7 months prior to use. Additionally, the DAP solidwas tested by differential scanning calorimetry (DSC) by heating from−20° C. to 200° C., back to −20° C. and again to 200° C.

Results

DAP powder was stable at 4° C. for more than 12 months. The results ofDSC are shown in FIG. 1. The second heat (“2”) showed similar thermalbehavior to the first heat (“1”), indicating that DAP survived repeatedheating to 200° C. This finding indicates that DAP is able to surviveheating to temperatures above those necessary for sterilization.

Example 2: Binding of DAP to Heparin and LMWH Materials and Methods

Dynamic light scattering (DLS) was used to assess association of 1 mg/mlunfractionated heparin and 1 mg/ml bemiparin (LMWH; HIBOR®), eitheralone or in combination with 100 mg/ml DAP in water (mass ratios of100:1 of DAP to heparin or LMWH).

Results

DAP physically associated in water with both unfractionated heparin(FIG. 2) and LMWH (not shown) to form physical associations thatincrease the apparent diameter. When solutions of DAP were mixed withsolutions of LMWH or unfractionated heparin, they formed particles dueto their physical interactions, which supports the theory that DAP couldreverse heparin and LMWH anticoagulation by physically associating withthese molecules.

Example 3: DAP Binding to Anticoagulants as Measured by DLS Materialsand Methods

Rivaroxaban (XARELTO®) alone, DAP alone, and DAP:rivaroxabancombinations at mass ratios of 1:1 and 10:1 were added into an aqueoussolution and analyzed by dynamic light scattering (DLS) to assessassociation of the DAP and rivaroxaban. A similar experiment wasconducted on apixaban (ELIQUIS®) alone, DAP alone, and DAP:apixabancombinations at mass ratios of 1:1, 10:1 and 100:1. Fondaparinux(ARIXTRA®) alone, DAP alone, and fondaparinux:DAP combinations at massratios of 1:1, 10:1 and 100:1 were similarly tested. LMWH (bemiparin;HIBOR®), alone, DAP alone, and LMWH:DAP combinations at mass ratios of1:1, 10:1, and 100:1 were also tested. The concentration of LMWH testedwas 0.1 mg/ml. Therefore, at 1:1, 0.1 mg/ml DAP was tested, at 10:1, 1mg/ml was tested, and at 100:1, 10 mg/ml DAP was tested.

Additionally, dabigatran alone, DAP alone, and dabigatran:DAPcombination at mass ratios of 1:1, 10:1, 100:1, 1,000:1, and 10,000:1DAP were tested. Finally, ARGATROBAN® alone, DAP alone, or combinationsof argatroban:DAP at mass ratios of 1:1, 10:1, 100:1, and 1,000:1 weretested.

Results

The results are shown in FIG. 3 for rivaroxaban; FIG. 4 for apixaban;FIG. 5 for fondaparinux (ARIXTRA®), FIG. 6 for LMWH; and FIG. 7 forargatroban. Each figure shows individual peaks representing DAP and theanticoagulant alone in aqueous solution. When the anticoagulant wasmixed with DAP at sufficiently high mass ratios, a change in size wasobserved. In this assay, even a slight increase in size indicatesphysical interaction between the two; however, only significant shiftsin the apparent diameter are used in assessing the in vitro in vivocorrelation. Apparent diameter is a measure of the degree ofinteraction.

Example 4: DAP Reversal of LMWH Anticoagulation In Vivo Materials andMethods

A male albino rat, weighing 470 g, was administered 10 mg of bemiparin(an overdose of LMWH) by subcutaneous injection. aPTT time was measuredover the course of five hours. Four hours after LMWH administration, therat received an intravenous dose of 200 mg/kg of DAP (100 mg DAP).

Results

Upon administration of LMWH, the aPTT rose from 53 to 246 seconds overthe course of four hours. Intravenous administration of 200 mg/kg of DAP(100 mg DAP) brought aPTT time below baseline within 60 minutes (FIG.8).

Example 5: DAP Reversal of Dabigatran (PRADAXA®) Anticoagulation InVivo; an Overdose Study Materials and Methods

A male albino rat, weighing 430 g, was administered 40 mg/kg of PRADAXA®(20 mg PRADAXA®; overdose of PRADAXA®) by oral gavage.

Approximately 2 hours into PRADAXA® treatment, 200 mg/kg DAP (100 mgDAP) was administered as an intravenous bolus injection. Approximately 2hours later, the rat was administered a dose of 100 mg/kg of DAP (50 mgDAP). In another hour, the rat was administered another dose of 100mg/kg of DAP (50 mg DAP). aPTT was measured throughout the course of theentire treatment.

Results

The results are shown in FIGS. 9 and 13. 2 hours followingadministration of PRADAXA®, aPTT rose from 43 to 81 seconds, showingsignificant anti-coagulation. 100 mg of DAP was administered as anintravenous bolus injection, which brought aPTT down below baselinewithin 25 minutes. 2 hours later, aPTT had risen back to 79 seconds andthe rat was administered a dose of 50 mg of DAP. Within 30 minutes, aPTTwas brought down below baseline. Both times, within 60 minutes followingDAP administration, the aPTT levels had returned above baseline. Afterthe second dose of DAP, the aPTT rose to 53 seconds. A third dose ofDAP, 100 mg/kg of DAP (50 mg DAP), was administered intravenously andthe aPTT was dropped to baseline within 20 minutes. FIG. 13 demonstratesa similar experiment where, after 15.5 mg/kg administration of PRADAXA®,the aPTT returned to normal within about 30 minutes of initiation of 100mg/kg DAP treatment.

Example 6: DAP Reversal of Unfractionated Heparin (“UHF”)Anticoagulation In Vivo Materials and Methods

A male albino rat, weighing 515 g, was administered 10 mg/kg ofunfractionated heparin (5 mg UFH) by subcutaneous injection.

200 mg/kg of DAP (100 mg DAP) was administered as two intravenous bolusinjections after UFH administration. Subsequently, the rat wasadministered a dose of 400 mg/kg of DAP (200 mg of DAP). aPTT wasmeasured throughout the course of the entire treatment.

Results

As demonstrated in FIG. 10, the aPTT time rose significantly from 28 to102 seconds over the course of one hour after administration of heparin.100 mg of DAP was administered intravenously and it brought aPTT time to48 seconds in 20 minutes. Within 1 hour, aPTT rose to 120 seconds, thenanother 100 mg of DAP was administered intravenously. In 15 minutes, theaPTT was lowered to 47 seconds. Within 1 hour, aPTT rose to 96 seconds,then a dose of 200 mg of DAP was administered intravenously. 10 minutesafter, aPTT dropped to 33 seconds.

Example 7: DAP Reversal of Rivaroxaban (XARELTO®) Anticoagulation InVivo Materials and Methods

5 mg/kg rivaroxaban (XARELTO®) was orally administered to rats. Afterfour hours, 5 mg/kg of DAP (2 mg DAP) was administered intravenously.aPTT were measured at zero, 15, 30, 45, 60 and 240 minutes, prior toadministration of DAP. aPTT was again measured at about 5, 10, 25, 35,45, 60, 120, and 240 minutes after DAP administration.

Results

The results are shown in FIG. 11. DAP effectively reversed therivaroxaban (XARELTO®) anticoagulation in vivo in rats.

Example 8: DAP Reversal of Fondaparinux (ARIXTRA®) Anticoagulation InVivo Materials and Methods

5 mg/kg fondaparinux was administered subcutaneously to rats. 200 mg/kgDAP was administered intravenously after 2 hours. Activity was measuredby chromogenic 510 k cleared Factor Xa Assay (Biophen) at 10, 20, 30 and60 minutes after DAP administration.

Results

FIG. 12 demonstrates DAP-mediated reversal of fondaparinuxanticoagulation within 10 minutes of administration.

Example 9: Intravenous DAP does not Influence aPTT Materials and Methods

0, 2, 10, 25, 50 or 100 mg DAP were administered intravenously to male,weight matched CD rats and aPTT was measured.

Results

The results shown in FIG. 16 demonstrate that DAP administeredintravenously did not influence aPTT in a dose dependent fashion in theabsence of anticoagulants. Error bars represent standard error fromseven aPTT measurements averaged over 90 minutes.

Example 10: DAP Reversal of Anticoagulation in a Rat Tail TransectionModel Materials and Methods

Three rats each were administered 2 mg of rivaroxaban. One rat receiveda sham reversal containing no DAP, the second received 2.5 mg of DAP,and the third received 12.5 mg DAP. A fourth fat received shamanticoagulant and reversal doses (“sham”). 20 minutes after the reversaldose, tails were transected 1 mm from the tip, placed in roomtemperature saline, and blood loss was collected for 30 minutes and thenweighed.

Same procedures were used with 1.25 mg apixaban (ELIQUIS®) alone or incombination with 5 or 12.5 mg DAP; with 15.5 mg dabigatran etexilate(PRADAXA®) alone or in combination with 5 or 12.5 mg DAP; and with 5 mgedoxaban (LIXIANA®) alone or in combination with 12.5 mg DAP.

Results

The results are shown in FIG. 15 for rivaroxaban, in FIG. 16 forapixaban, in FIG. 17 for edoxaban, and in FIG. 18 for dabigatranetexilate. The rat tail transection bleeding assay is analogous to theclinical situation in which acute anticoagulant reversal is needed.Results show that DAP effectively reversed anticoagulant activityleading to statistically significant reduction in blood loss compared torats receiving anticoagulant only.

Example 11: DAP Reversal of Rivaroxaban (XARELTO®) Anticoagulation inFreshly Drawn Human Blood Ex Vivo Materials and Methods

Human blood was drawn from a volunteer. Rivaroxaban at 0.25 μg/ml wasadded alone or in combination with 50 μg/ml DAP. Controls contained 50μg/ml DAP or saline. aPTT was measured within 2 minutes of bloodcollection.

Results

FIG. 19 demonstrates that administration of DAP led to a reversal ofrivaroxaban-induced anticoagulation in freshly drawn human blood, asmeasured by aPTT. Error bars represent standard error from threeindependent experiments.

Example 12: DAP Reversal of Rivaroxaban and Apixaban Anticoagulation inHuman Plasma Ex Vivo Materials and Methods

218 μg/L or 459 μg/L of rivaroxaban was added to human plasma, with orwithout 1,250 μg/L or 6,250 μg/L of DAP, respectively. Similarly, 156μg/L or 313 μg/L of apixaban was added to human plasma with or without1,156 μg/L or 3,125 μg/L of DAP, respectively. DAP effect onanticoagulation was measured by 510 k cleared Biophen anti-Factor Xachromogenic assay. Rivaroxaban concentrations were determined bycomparison with plasma calibration standards, while apixabanconcentrations were inferred from stock solution dilutions ascalibration standards are not yet available.

Results

For both concentrations of rivaroxaban and apixaban, DAP returned theeffective anticoagulant concentration to below the minimum effectiveconcentration. FIG. 20 shows the results for rivaroxaban and FIG. 21shows the results for apixaban.

Example 13: DAP Dose-Dependent Reversal of Rivaroxaban Anticoagulationin Human Plasma Ex Vivo Materials and Methods

218 μg/L rivaroxaban was added to human plasma either alone or incombination with 1.25, 12.5, 125, or 1,250 μg/L of DAP. Factor Xaactivity was measured by 510 k cleared Biophen anti-Xa chromogenic assaykit. Rivaroxaban concentrations were determined by comparison withplasma calibration standards.

Results

FIG. 22 demonstrates that DAP was effective in dose-dependent reversalof rivaroxaban anticoagulation in human plasma, as demonstrated by itseffect on rivaroxaban concentration (measured by Factor Xa activityassay).

1. A compound of formula IY-M-X-L-A-L′-X′-M′-Y′  (I) or a pharmaceutically acceptable saltthereof, wherein: A is a substituted or unsubstituted aromatic ornon-aromatic, carbocyclic or heterocyclic ring or a linear moiety; L andL′ are the same or different and are linkers; X and X′ are the same ordifferent and are absent or are a functional group that attaches thelinker L to M and linker L′ to M′, respectively; M and M′ are the sameor different and are absent or is a linker that attaches X to Y and X′to Y′, respectively; and Y and Y′ are the same or different and are amoiety containing one or more cationic atoms or groups or one or moregroups that become cationic under physiological conditions.
 2. Thecompound of claim 1, wherein A is a non-aromatic, heterocyclic ring or alinear moiety, and wherein the ring or the linear moiety containreactive functional groups that can form a bond to X and/or X′, whenpresent, or Y and/or Y′.
 3. The compound of claim 2, wherein A is apiperazine or diketopiperazine.
 4. The compound of claim 1, wherein Xand X′, when present, is a functional group that attaches linker L to Yand linker L′ to Y′, respectively, and wherein the functional group isselected from the group consisting of esters, amides, and ketones. 5.The compound of claim 1, wherein L and/or L′ is a substituted orunsubstituted alkylene chain that is C₁ to C₆.
 6. The compound of claim1, wherein the compound is represented by formula II:

or a pharmaceutically acceptable salt thereof.
 7. The compound of claim6, wherein the compound is represented by formula III:

or a pharmaceutically acceptable salt thereof.
 8. The compound of claim7, wherein the compound is represented by formula IV:

or a pharmaceutically acceptable salt thereof, wherein n is 3 to 5, m is3 to 6 and G is selected from —NH₂ and OH.
 9. The compound of claim 8,wherein G is amino.
 10. The compound of claim 1, wherein Y and Y′ areindependently selected from


11. The compound of claim 10, wherein Y and Y′ is


12. A compound of formula V

or a pharmaceutically acceptable salt thereof.
 13. A compound of formulaVI

or a pharmaceutically acceptable salt thereof.
 14. A compound of formulaVII

or a pharmaceutically acceptable salt thereof.
 15. A compound of formulaVIII

or a pharmaceutically acceptable salt thereof.
 16. A pharmaceuticalcomposition comprising the compound of claim 1 and a pharmaceuticallyacceptable carrier.
 17. The pharmaceutical composition of claim 16,wherein the composition is adapted for enteral administration.
 18. Thepharmaceutical composition of claim 17, wherein the composition isadapted for oral administration.
 19. The pharmaceutical composition ofclaim 16, wherein the composition is adapted for parenteraladministration.
 20. The pharmaceutical composition of claim 19, whereinthe composition is adapted for intravenous or subcutaneousadministration.
 21. A method of completely or partially reversing ananticoagulant effect of a coagulation inhibitor comprising administeringto a subject in need thereof a therapeutically effective amount of acompound of claim 1 or pharmaceutically acceptable salt thereof.
 22. Themethod of claim 21, wherein the coagulation inhibitor is selected fromthe group consisting of an unfractionated heparin, low molecular weightheparin (LMWH), Factor Ha inhibitor, and Factor Xa inhibitor.
 23. Themethod of claim 22, wherein the coagulation inhibitor is a factor Xainhibitor.
 24. The method of claim 23, wherein the factor Xa inhibitoris selected from the group consisting of rivaroxaban, apixaban,edoxaban, and fondaparinux.
 25. The method of claim 21, wherein thesubject is a mammal.
 26. The method of claim 25, wherein the subject isa human.
 27. The method of claim 21, wherein the complete or partialreversal of an anticoagulant effect of a coagulation inhibitor ismeasured by anti-factor Xa activity assay.
 28. The method of claim 21,wherein the subject in need thereof is a subject in whom acute orplanned reversal of anticoagulation is indicated.
 29. The method ofclaim 28, wherein the subject in whom acute or planned reversal ofanticoagulation is indicated is a subject suffering from anticoagulantoverdose, a subject suffering from hemorrhage, a subject requiringplanned surgical intervention, a subject undergoing an invasive ornon-invasive procedure requiring a biopsy, a subject undergoing aprocedure wherein a procedural error may result in hemorrhage if thesubject remains anticoagulated, or a subject requiring spinal orepidural anesthesia.
 30. The method of claim 28, wherein the subject inneed thereof is a subject receiving anticoagulation for strokeprevention, cardiac surgical and diagnostic procedures, cardiacarrhythmias, deep vein thrombosis (DVT) prevention, pulmonary embolism,or generally for the prevention of pathologic blood clots.
 31. Themethod of claim 22, wherein the coagulation inhibitor is a LMWH, andwherein the LMWH is selected from the group consisting of bemiparin,certoparin, dalteparin, enoxaparin, nadroparin, parnaparin, reviparin,and tinzaparin.
 32. The method of claim 21, wherein the compound or thepharmaceutically acceptable salt thereof is administered at a dose massratio of between about 0.01:1 to about 1000:1 of the compound or thepharmaceutically acceptable salt thereof to anticoagulant.
 33. Themethod of claim 32, wherein the compound or the pharmaceuticallyacceptable salt thereof is administered at a dose mass ratio of about10:1 of the compound or the pharmaceutically acceptable salt thereof toanticoagulant.
 34. The method of claim 21, wherein the method comprisesadministering at least one additional therapeutic agent.
 35. The methodof claim 34, wherein the at least one additional therapeutic agent isvitamin K.
 36. A diagnostic kit comprising the compound of claim
 1. 37.The kit of claim 36, wherein the kit is used for determining ananticoagulant concentration in blood.